PROJECT SUMMARY Hot-spot mutations in the isocitrate dehydrogenase 1 (IDH1) gene are highly recurrent in lower grade gliomas (LGGs) and secondary glioblastomas (GBMs) and encode the mutant IDH1-R132H enzyme. This enzyme displays neomorphic activity, converting 2-oxoglutarate (2OG) to the oncometabolite (R)-2-hydroxyglutarate ((R)-2HG). (R)-2HG accumulates to millimolar levels in IDH1 mutant gliomas and promotes gliomagenesis by competitively modulating 2OG-dependent enzymes that regulate glial cell transformation. Although these discoveries have profoundly reshaped our understanding of the molecular pathogenesis of these diseases, these insights have not yet translated to the development of new, effective therapeutic strategies for glioma patients. Direct inhibitors of the mutant IDH enzyme have shown poor activity in early preclinical and clinical studies of IDH mutant glioma relative to the robust efficacy they display against IDH mutant leukemias. These findings highlight the clinical need for alternative therapeutic strategies to treat IDH1 mutant gliomas. I hypothesize that novel treatment strategies can be developed by exploiting synthetic lethality with the IDH1- R132H oncogene. I performed a chemical synthetic lethality screen using isogenic IDH1 mutant and wild-type glioma cell lines and found that multiple pyrimidine synthesis inhibitors preferentially killed IDH1 mutant cells. My preliminary data shows that (R)-2HG-mediated inhibition of the 2OG-dependent branched chain amino acid transaminases BCAT1 and BCAT2 impairs nitrogen incorporation into the pyrimidine biosynthesis pathway. Therefore, in Aim 1 I will test whether reduced BCAT activity represents the molecular mechanism underlying mutant IDH1-induced pyrimidine synthesis inhibitor hypersensitivity. I will go on to assess whether the IDH1- R132H mutation is a predictive biomarker for response to pyrimidine synthesis inhibitors by testing the cytotoxicity of these agents against a panel of IDH1 mutant and wild-type glioma neurosphere lines. Finally, I will test a novel brain-penetrant inhibitor of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) alone and in combination with radiotherapy in xenograft models of IDH1 mutant glioma. Another major impediment to the development of new, effective therapeutic strategies for IDH1 mutant glioma is a paucity of genetically-engineered mouse (GEM) models of this disease that can be used to evaluate such strategies. In Aim 2 I propose to establish the first Crispr/Cas9-based GEM models of glioma driven by mutant IDH1. I have successfully produced mutant Idh1-driven Grade III anaplastic astrocytomas in mice and I will optimize my approach to increase astrocytoma penetrance in this GEM model. I will also modify my strategy to generate isogenic GEM models of IDH1 mutant and wild-type GBM that can be used for future in vivo studies of synthetic lethality with the Idh1-R132H oncogene. If successful, these projects will establish rationale for clinical testing of pyrimidine synthesis inhibitors for IDH1 mutant glioma therapy and generate GEM models of this disease to support future testing of newly devised treatment strategies.